242 Florida Entomologist 96(1) March 2013 A NEW INVASIVE JACK BEARDSLEY MEALYBUG, PSEUDOCOCCUS JACKBEARDSLEYI (HEMIPTERA: PSEUDOCOCCIDAE) ON PAPAYA IN INDIA M. MANI1, SUNIL JOSHI2, M. KALYANASUNDARAM3, C. SHIVARAJU1, A. KRISHNAMOORTHY1, R. ASOKAN4 AND K. B. REBIJITH4,* 1Division of Entomology & Nematology, 4Division of Biotechnology, Indian Institute of Horticulture Research, Hessaraghatta Lake post, Bangalore-560089, India 2National Bureau of Agriculturally Important Insects, Bangalore-560024, India 3Tamil Nadu Agricultural University, Coimbatore-641003, India *Corresponding author; Email: [email protected] Mealybugs (Hemiptera: Pseudococcidae) trilocular pores on sclerotised area. All other cera- are major pests of a wide range of agricultural, rii on a membranous area. Anterior cerari with horticultural and ornamental plants worldwide relatively smaller setae and with 2 to 3 auxiliary (Miller et al. 2002). Certain of attributes of the seate. Cerarii number C7 and cerarii on head with Pseudococcidae, viz., wide host range, short 3-4 conical setae. Dorsal surface with small se- generation time, cosmopolitan nature, ability to ate and trilocular pores scattered almost evenly. transmit some important plant viruses, etc., have All frontal cerarri with oral rim tubular duct just contributed to their enormous damage potential behind them. Other oral rim tubular ducts pres- (Meyer et al. 2008). In this regard, Pseudococcus ent submedially and submarginally on thorax jackbeardsleyi Gimpel and Miller (Hemiptera: and abdomen. Anal lobe and penultimate cerari Pseudococcidae), known as the Jack Beardsley with oral collar tubular duct between them. Ven- mealybug, is a polyphagous species of neotropi- tral surface with slender setae. Multilocular disc cal origin that is known to attack 93 plant spe- pores present on V to VII abdominal segments. cies including several vegetable and fruit and Oral rim tubular duct and oral collar tubular ornamental crop species (CAB Intl. 2001). The ducts same as dorsum. (Fig. 1). earliest record of this mealybug in Asia was from The invasive mealybug is grayish in color; thin Singapore in 1958, followed by Malaysia in 1969, filaments around the body, caudal pair about one Indonesia in 1973, Philippines in 1975, Brunei in half of the length of the body, and ovisac cover- 1979, Thailand in 1987, the Maldives, and Viet- ing hind part of the body. All other morphological nam in 1994 (Williams 2004a,b; Muniappan 2011) characters also match the description given by and not reported yet from India. In the current re- Williams (2004a). Quick and correct identifica- port during the survey for monitoring the activity tion of an invasive pest like P. jackbeardsleyi is of papaya mealybug, Paracoccus marginatus Wil- important in order to initiate suitable manage- liams and Granara de Willink and its parasitoid ment measures that may include quarantine. Mo- Acerophagus papayae Noyes & Schauff in Satya- lecular identification, at this juncture is handy as mangalam (N 11.5200° E 77.2500°) Tamilnadu, it is not limited by life stage, polymorphism and India, a short tailed mealybug and P. marginatus gender. Incursion of this pest into India has been were found colonizing 2 papaya plantations on proven so far by identifying it by morphological or Jun 2012. Mealybugs were found scattered on molecular methods. Hence, in this paper we are the leaves, fruits and trunks and were observed providing evidence that identifies P. jackbeards- sucking the sap from various parts of the papaya leyi in India by both morphological and molecular plants including leaves, stems, flowers and fruits. methods. No serious damage was observed on the papaya Standard protocols were followed for DNA ex- plant caused by this new mealybug. Specimens traction, Polymerase Chain Reaction, sequencing of this mealybug were collected and preserved in of the mitochondrial cytochrome oxidase I (CO-I) ethyl alcohol (100%) and were stored at 20 °C un- gene fragment and sequence alignment (Hajiba- til further use. The mealybug was identified as baei et al. 2005). Small portion of the abdomen the Jack Beardsley mealybug, P. jackbeardsleyi, from individual P. jackbeardsleyi was used for at the National Bureau of Agriculturally Impor- genomic DNA and PCR was carried out in a ther- tant Insects, Bangalore. mal cycler (ABI-Applied Biosystems, Veriti, USA) Morphological characters used for identify- with the following cycling parameters; 94 °C for 4 ing P. jackbeardsleyi are as follows: Body of the min as initial denaturation followed by 35 cycles female oval to elongate. Antenna 8 segmented. of 94 °C for 30 s, 47 °C for 45 s, 72 °C for 45 s and Legs well developed, translucent pores present on 72 °C for 20 min as final extension using univer- hind femur and hind tibia. Discoidal pores pres- sal CO-I primers (LCO-1490- 5’-GGT CAA CAA ent near eye on sclerotised rim. Cerarii 17 pairs. ATC ATA AAG ATA TTG G-3’ and HCO-2198- 5’- Anal lobe cerari with 2 conical setae and many TAA ACT TCA GGG TGA CCA AAA AAT CA-3’, Scientific Notes 243 Fig 1. Morphological characters used for identification ofPseudococcus jackbeardsleyi Gimpel and Miller, col- lected on papaya from Satyamangalam,Tamilnadu. Hebert et al. 2004). PCR was performed in 25-µL cloning vector, InsT/Aclone and transformation total reaction volume containing 20 picomoles of using Escherichia coli (DH5α) cells was carried each primer, 10 mM Tris HCl (pH-8.3), 50 mM out (Fermentas GmBH, Germany) according to KCl, 2.5 mM MgCl2, 0.25 mM of each dNTP and the manufacturer’s protocol. Blue/white selection 0.5 U of Taq DNA polymerase (Fermentas Life was carried out and all the white colonies (colo- Sciences, EU). The voucher specimen deposited nies harbouring the insert) were maintained on in the National Pusa Collection, (NPC), New LBA containing ampicillin (100 mg/mL), incubat- Delhi. The amplified products were resolved in ed at 37 °C overnight and stored at 4 °C until fur- 1.0% agarose gel, stained with ethidium bromide ther use. Plasmids were isolated by GeneJET™ (10 µg/mL) and visualized in a gel documenta- Plasmid Miniprep Kit (Fermentas, Germany) tion system (UVP). The PCR amplified frag- according to manufacturer’s protocol, from over- ments were eluted using Nucleospin® Extract night cultures of the 5 randomly selected clones II according to the manufacturer’s protocol (MN, multiplied in LB broth. Sequencing was carried Germany) and ligated into the general purpose- out in an automated sequencer (ABI Prism 310; 244 Florida Entomologist 96(1) March 2013 Applied Biosystems, USA) using M13 universal similarity with the other 3 accessions found in primers both in forward and reverse directions. the BLAST search and used for constructing the Homology search was carried out using BLAST phylogenetic tree. (http://www.ncbi.nlm.nih.gov) and the differences As many as 22 plant species have been report- in CO-I sequences of P. jackbeardsleyi were de- ed as hosts of P. jackbeardsleyi in Asian countries. termined using the sequence alignment editor There is no record of this mealybug on papaya in ‘Bioedit’. A sequence for P. jackbeardsleyi was de- south Asian countries (Williams 2004a,b). Thus, posited with the NCBI database, and the acces- we report P. jackbeardsleyi for the first time in sion number is KC119455. Sequence generated in India and for the first time breeding on papaya. the present study along with the other Pseudococ- No classical biological control attempt has cus spp. viz. Pseudococcus comstocki (Kuwana), been made against the Jack Beardley mealy- Pseudococcus viburni (Signoret) and Ferrisia vir- bug, and possibly it is kept under control by the gata (Cockerell) (as an outgroup) (Retrieved from local natural enemies in certain invaded coun- NCBI) were aligned using BioEdit. 7. 0 program tries (Muniappan et al. 2011). Also we observed using Clustal W. that the coccinellid, Cryptolaemus montrouzieri Mitochondrial cytochrome oxidase-1 (CO-I) Mulsant, was capable of checking Jack Beard- was successfully sequenced from an individual ley mealybug populations in the present study. P. jackbeardsleyi. A comparison of the triplicate Hence there is no need for any panic to cope sequences showed no evidence of mismatch, in- with the newly invasive P. jackbeardsleyi in In- dicated there were no sequencing errors. A total dia. However, our study will aid in proper quar- fragment size of 647 bp of the CO-I was analyzed antine measures and emergency management for P. jackbeardsleyi. Evidence of nuclear copies plans by which further spread into other states was not found, which was supported by the ab- may be hindered or delayed to avoid economic sence of stop codons within the sequences and the losses. base composition was similar with no indels (Re- bijith et al. 2012). Ferrisia virgata was selected SUMMARY as an out group. Phylogenetic analysis of aligned sequences was done using MEGA. 5. 0 (Tamura Mealybugs are major factors limiting the pro- et al. 2011). The method of neighbor-joining (NJ) ductivity of papaya in India. In June 2012 dur- with the Kimura two-parameter model (Kimu- ing a survey for the papaya mealybug, Paracoc- ra 1980) was selected to build the phylogenetic cus marginatus, and its parasitoid Acerophagus tree. To assess the robustness of the tree, 1000 papayae, in Satyamangalam, Tamilnadu, India, bootstrap replicates were selected. Pseudococcus a short tailed mealybug was found together with jackbeardsleyi showed 8.0% (52/649) and 8.1% P. marginatus colonizing papaya in 2 plantations. (53/649) sequence variation with Pseudococcus This mealybug was identified as the Jack Beards- comstocki and Pseudococcus viburni respectively. ley mealybug, P. jackbeardsleyi Gimpel and Mill- Phylogenetic tree for sequences of all Pseudococ- er (Hemiptera: Pseudococcidae), by an integrated cus spp. used in the present study are shown in taxonomic approach. This work appears to be the Fig. 2. This figure and the sequences suggest that first report ofP. jackbeardsleyi in India and of pa- P.